Ductal lavage for detection of cellular atypia in women at high risk for breast cancer.

BACKGROUND: Breast cancer originates in breast epithelium and is associated with progressive molecular and morphologic changes. Women with atypical breast ductal epithelial cells have an increased relative risk of breast cancer. In this study, ductal lavage, a new procedure for collecting ductal cells with a microcatheter, was compared with nipple aspiration with regard to safety, tolerability, and the ability to detect abnormal breast epithelial cells. METHODS: Women at high risk for breast cancer who had nonsuspicious mammograms and clinical breast examinations underwent nipple aspiration followed by lavage of fluid-yielding ducts. All statistical tests were two-sided. RESULTS: The 507 women enrolled included 291 (57%) with a history of breast cancer and 199 (39%) with a 5-year Gail risk for breast cancer of 1.7% or more. Nipple aspirate fluid (NAF) samples were evaluated cytologically for 417 women, and ductal lavage samples were evaluated for 383 women. Adequate samples for diagnosis were collected from 111 (27%) and 299 (78%) women, respectively. A median of 13,500 epithelial cells per duct (range, 43-492,000 cells) was collected by ductal lavage compared with a median of 120 epithelial cells per breast (range, 10-74,300) collected by nipple aspiration. For ductal lavage, 92 (24%) subjects had abnormal cells that were mildly (17%) or markedly (6%) atypical or malignant (<1%). For NAF, corresponding percentages were 6%, 3%, and fewer than 1%. Ductal lavage detected abnormal intraductal breast cells 3.2 times more often than nipple aspiration (79 versus 25 breasts; McNemar's test, P<.001). No serious procedure-related adverse events were reported. CONCLUSIONS: Large numbers of ductal cells can be collected by ductal lavage to detect atypical cellular changes within the breast. Ductal lavage is a safe and well-tolerated procedure and is a more sensitive method of detecting cellular atypia than nipple aspiration.

Feline Pit2 functions as a receptor for subgroup B feline leukemia viruses.

Different subgroups of feline leukemia virus (FeLV) use different host cell receptors for entry. Subgroup A FeLV (FeLV-A) is the virus that is transmitted from cat to cat, suggesting that cells expressing the FeLV-A receptor are important targets at the earliest stages of infection. FeLV-B evolves from FeLV-A in the infected cat through acquisition of cellular sequences that are related to the FeLV envelope gene. FeLV-Bs have been shown to infect cells using the Pit1 receptor, and some variants can infect cells at a lower efficiency using Pit2. Because these observations were made using receptor proteins of human or rodent origin, the role that Pit1 and Pit2 may play in FeLV-B replication in the cat is unclear. In this study, the feline Pit receptors were cloned and tested for their ability to act as receptors for different FeLV-Bs. Some FeLV-Bs infected cells expressing feline Pit2 and feline Pit1 with equal high efficiency. Variable region A (VRA) in the putative receptor-binding domain (RBD) was a critical determinant for both feline Pit1 and feline Pit2 binding, although other domains in the RBD appear to influence how efficiently the FeLV-B surface unit can bind to feline Pit2 and promote entry via this receptor. An arginine residue at position 73 in VRA was found to be important for envelope binding to feline Pit2 but not feline Pit1. Interestingly, this arginine is not found in endogenous FeLV sequences or in recombinant viruses recovered from feline cells infected with FeLV-A. Thus, while FeLV-Bs that are able to use feline Pit2 can evolve by recombination with endogenous sequences, a subsequent point mutation during reverse transcription may be needed to generate a virus that can efficiently enter the cells using the feline Pit2 as its receptor. These studies suggest that cells expressing the feline Pit2 protein are likely to be targets for FeLV-B infection in the cat.

Specificity in receptor usage by T-cell-tropic feline leukemia viruses: implications for the in vivo tropism of immunodeficiency-inducing variants.

Cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T) evolve from FeLV-A in infected animals and demonstrate host cell specificities that are distinct from those of their parent viruses. We recently identified two cellular proteins, FeLIX and Pit1, required for productive infection by these immunodeficiency-inducing FeLV-T variants (M. M. Anderson, A. S. Lauring, C. C. Burns, and J. Overbaugh, Science 287:1828-1830, 2000). FeLV-T is the first example of a naturally occurring type C retrovirus that requires two proteins to gain entry into target cells. FeLIX is an endogenous protein that is highly related to the N-terminal portion of the FeLV envelope protein, which includes the receptor-binding domain. Pit1 is a multiple-transmembrane phosphate transport protein that also functions as a receptor for FeLV-B. The FeLV-B envelope gene is derived by recombination with endogenous FeLV-like sequences, and its product can functionally substitute for FeLIX in facilitating entry through the Pit1 receptor. In the present study, we tested other retrovirus envelope surface units (SUs) with their cognate receptors to determine whether they also could mediate infection by FeLV-T. Cells were engineered to coexpress the transmembrane form of the envelope proteins and their cognate receptors, or SU protein was added as a soluble protein to cells expressing the receptor. Of the FeLV, murine leukemia virus, and gibbon ape leukemia virus envelopes tested, we found that only those with receptor-binding domains derived from endogenous FeLV could render cells permissive for FeLV-T. We also found that there is a strong preference for Pit1 as the transmembrane receptor. Specifically, FeLV-B SUs could efficiently mediate infection of cells expressing the Pit1 receptor but could only inefficiently mediate infection of cells expressing the Pit2 receptor, even though these SUs are able to bind to Pit2. Expression analysis of feline Pit1 and FeLIX suggests that FeLIX is likely the primary determinant of FeLV-T tropism. These results are discussed in terms of current models for retrovirus entry and the interrelationship among FeLV variants that evolve in vivo.

Identification of envelope determinants of feline leukemia virus subgroup B that permit infection and gene transfer to cells expressing human Pit1 or Pit2.

The retroviral vector systems that are in common use for gene therapy are designed to infect cells expressing either of two widely expressed phosphate transporter proteins, Pit1 or Pit2. Subgroup B feline leukemia viruses (FeLV-Bs) use the gibbon ape leukemia virus receptor, Pit1, as a receptor for entry. Our previous studies showed that some chimeric envelope proteins encoding portions of FeLV-B could also enter cells by using a related receptor protein, Pit2, which serves as the amphotropic murine leukemia virus receptor (S. Boomer, M. Eiden, C. C. Burns, and J. Overbaugh, J. Virol. 71:8116--8123, 1997). Here we show that an arginine at position 73 within variable region A (VRA) of the FeLV-B envelope surface unit (SU) is necessary for viral entry into cells via the human Pit2 receptor. However, C-terminal SU sequences have a dominant effect in determining human Pit2 entry, even though this portion of the protein is outside known receptor binding domains. This suggests that a combination of specific VRA sequences and C-terminal sequences may influence interactions between FeLV-B SU and the human Pit2 receptor. Binding studies suggest that the C-terminal sequences may affect a postbinding step in viral entry via the Pit2 receptor, although in all cases, binding of FeLV-B SU to human Pit2 was weak. In contrast, neither the arginine 73 nor specific C-terminal sequences are required for efficient binding or infection with Pit1. Taken together, these data suggest that different residues in SU may interact with these two receptors. The specific FeLV-Bs described here, which can enter cells using either human Pit receptor, may be useful as envelope pseudotypes for viruses used in gene therapy.

Identification of a cellular cofactor required for infection by feline leukemia virus.

Retroviral infection involves continued genetic variation, leading to phenotypic and immunological selection for more fit virus variants in the host. For retroviruses that cause immunodeficiency, pathogenesis is linked to the emergence of T cell-tropic, cytopathic viruses. Here we show that an immunodeficiency-inducing, T cell-tropic feline leukemia virus (FeLV) has evolved such that it cannot infect cells unless both a classic multiple membrane-spanning receptor molecule (Pit1) and a second coreceptor or entry factor are present. This second receptor component, which we call FeLIX, was identified as an endogenously expressed protein that is similar to a portion of the FeLV envelope protein. This cellular protein can function either as a transmembrane protein or as a soluble component to facilitate infection.

Cloning of the cellular receptor for feline leukemia virus subgroup C (FeLV-C), a retrovirus that induces red cell aplasia.

Feline leukemia virus-C (FeLV-C) causes red cell aplasia in cats, likely through its interaction with its cell surface receptor. We identified this receptor by the functional screening of a library of complementary DNAs (cDNA) from feline T cells. The library, which was cloned into a retroviral vector, was introduced into FeLV-C-resistant murine (NIH 3T3) cells. The gene conferring susceptibility to FeLV-C was isolated and reintroduced into the same cell type, as well as into FeLV-C-resistant rat (NRK 52E) cells, to verify its role in viral infection. The receptor cDNA is predicted to encode a protein of 560 amino acids with 12 membrane-spanning domains, termed FLVCR. FLVCR has significant amino acid sequence homology with members of the major facilitator superfamily and especially D-glucarate transporters described in bacteria and in C. elegans. As FeLV-C impairs the in vivo differentiation of burst-forming unit-erythroid to colony-forming unit-erythroid, we hypothesize that this transporter system could have an essential role in early erythropoiesis. In further studies, a 6-kb fragment of the human FLVCR gene was amplified by polymerase chain reaction from genomic DNA, using homologous cDNA sequences identified in the human Expressed Sequence Tags database. By radiation hybrid mapping, the human gene was localized to a 0.5-centiMorgan region on the long arm of chromosome 1 at q31.3.

Leukemic phase of mantle cell lymphoma, blastoid variant.

Six patients with mantle cell lymphoma, blastoid variant, involving the blood are described. The circulating blast-like cells suggested the possibility of acute leukemias, chronic lymphoproliferative disorders or peripheralized lymphomas. The WBC counts ranged from 3,700 to 249,000/microL (3.7-249.0 x 10(9)/L) and the absolute lymphocyte counts from 1,000 to more than 200,000/microL (1.0 to > 200.0 x 10(9)/L). The peripheral blood smears showed a spectrum of cells, from small mature lymphocytes with irregular nuclei to medium-sized lymphocytes with blast-like chromatin. However, the morphologic features in a lymph node biopsy specimen and the immunophenotype confirmed a diagnosis of mantle cell lymphoma, blastoid variant. By flow cytometry the lymphoma cells expressed B-cell-associated antigens (CD19, CD20 and CD22), coexpressed CD5, lacked CD23, and expressed moderate intensity monoclonal surface immunoglobulin and CD20. Cytogenetic analysis showed the characteristic t(11;14) in 2 of 4 analyzed specimens. Mantle cell lymphoma, blastoid variant, is part of the differential diagnosis for blast-like cells.

Positive smoking history as a preliminary screening device for substance use in pregnant adolescents.

STUDY OBJECTIVE: To assess the utility of a history of smoking as a clinical indicator of substance use in pregnant adolescents. DESIGN: A secondary analysis of a subsample of all 15- to 19-year-old live-birth mothers (N = 1640) taken from the 1988 National Maternal and Infant Health Survey (NMIHS). Self-reported data were weighted and analyzed covering ethnicity, substance use, socioeconomic status, and prenatal care. Contingency table analysis and stepwise logistic regression were applied to compare the prevalence of other forms of substance use among smokers versus nonsmokers, and to evaluate whether a history of smoking made a unique contribution to identifying adolescents at increased risk for use of other substances. The specific substances studied were alcohol and cocaine which are known to be important contributors to perinatal morbidity and mortality. RESULTS: In this multiethnic, nationally representative subsample of pregnant adolescents, 35% reported a positive history of tobacco use 12 months before delivery. Another 32% had consumed alcohol, 9% had used marijuana, and about 1.5% had reported some use of cocaine or crack. The findings show greater use of tobacco by whites, non-Hispanics, the ever married, and women receiving prenatal care in the private sector. Fifty-three percent of all admitted users of alcohol or cocaine smoked cigarettes 12 months before delivery. Logistic regression shows that smokers were four times more likely to use alcohol or cocaine than nonsmokers when controlling for other sociodemographic and economic variables. CONCLUSION: Substance use is common in pregnant adolescents from all ethnic and economic backgrounds. Self-report of smoking may be useful in screening for adolescents at risk for using cocaine or alcohol during pregnancy.

Ki-1 anaplastic large cell lymphoma with a prominent leukemic phase.

Patients with Ki-1 (CD30)-positive anaplastic large cell lymphoma (ALCL) occasionally have rare circulating lymphoma cells. We report a case of ALCL with a prominent leukemic phase. A 36-year-old man presented with widespread ALCL involving lymph nodes, spleen, pleural fluid, cerebrospinal fluid, and bone marrow. His white blood cell count was 106,000/mm3 with 20% lymphoma cells. The circulating neoplastic cells were large, and had irregular nuclei with multiple small nucleoli, ample cytoplasm, and multiple clear cytoplasmic vacuoles. This case illustrates that ALCL may have a marked leukemic phase composed of large anaplastic cells.

Activation of the human thymidine kinase (TK) promoter by simian virus 40 large T antigen requires both the T antigen pRb family-binding domain and TK promoter sequences resembling E2F-binding sites.

Infection of quiescent cells with the DNA tumor virus simian virus 40 induces expression of the cellular thymidine kinase (TK) gene a minimum of 10- to 20-fold, and this induction depends upon the viral protein large T antigen (T-Ag). To define both human TK promoter elements and T-Ag functional domains required for transcriptional induction, we have established a system in which stable Rat-1 transfectants harboring TK promoter-luciferase hybrid genes are infected with recombinant adenoviruses expressing either wild-type or mutant forms of T-Ag and luciferase expression is measured as an indicator of promoter activity. The results show that (i) a 135-bp TK promoter fragment is activated 10- to 15-fold by viral infection; (ii) this activation is the result of both T-Ag-dependent and -independent mechanisms; (iii) the T-Ag pRb family-binding domain, but not the p53-binding, helicase, or ATPase domain, is required for activation; and (iv) activation is severely diminished with a TK promoter fragment in which E2F-like-binding sites have been removed. These data demonstrate a requirement for both an E2F-related factor and a pRb family member in activation of the TK promoter by T-Ag. This contrasts with the promiscuous activation of many cellular and viral genes by T-Ag, which is independent of its ability to bind pRb.

In vitro antiplasmodial, antiamoebic, and cytotoxic activities of a series of bisbenzylisoquinoline alkaloids.

Twenty-four bisbenzylisoquinoline alkaloids were screened for antiplasmoidal, antiamoebic, and cytotoxic activities by use of in vitro microtests. Eight of the alkaloids had antiplasmodial activity, with a 50% inhibitory concentration (IC50) of less than 1 microM against a multidrug-resistant strain of Plasmodium falciparum (chloroquine had an IC50 of 0.2 microM). The three alkaloids most active against Entamoeba histolytica, aromoline, isotrilobine, and insularine, had IC50s of 5 to 11.1 microM (metronidazole had an IC50 of 1.87 microM). None of the 24 bisbenzylisoquinoline alkaloids exhibited significant cytotoxicity against the KB cell line, the most toxic being berbamine, with an IC50 of 17.8 microM (the IC50 of podophyllotoxin was 0.008 microM). Bisbenzylisoquinoline alkaloids merit further investigation as potential novel antimalarial agents.

Frequency of ultraviolet radiation-induced mutation at the hprt locus in repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4.

The frequency of spontaneous and ultraviolet radiation (UVR)-induced mutation at the hprt locus was determined in control and denV-transfected, repair-proficient murine fibroblasts. Control cells removed an average of 25% of pyrimidine dimers induced by exposure to 150 J/m2 UVR from an FS40 sunlamp within 24 h; under the same conditions of induction and repair, denV-transfected cells removed an average of 71% of pyrimidine dimers. Control cells were somewhat more resistant than denV-transfected cells to killing by UVR. The average frequency of spontaneous mutation at the hprt locus for control and denV-transfected cells was 3 and 15 6-thioguanine (6-TG)-resistant colonies per 10(6) surviving cells, respectively; there was no statistically significant difference between control and denV-transfected cells. However, after exposure to 75 or 150 J/m2 UVR, denV-transfected cells had a significantly lower frequency of mutation to 6-TG resistance. After exposure to a fluence of 75 J/m2, the average frequency of UVR-induced mutation at the hprt locus was 166 mutant colonies per 10(6) surviving cells for control cells and 92 mutant colonies for denV-transfected cells; after 150 J/m2, control cells had 205 6-TG-resistant colonies per 10(6) cells, while denV-transfected cells had 61 mutant colonies. These results demonstrate that UVR-induced pyrimidine dimers are mutagenic photoproducts in mammalian cells.

Regulation of the cellular thymidine kinase gene promoter in simian virus 40-infected cells.

We examined the regulation of the cellular thymidine kinase (TK) gene promoter in simian virus 40 (SV40)-infected simian CV1 cells. Nuclear run-on transcription assays demonstrated a three- to fourfold increase in the rate of transcription of the endogenous gene at 14 to 16 h following viral infection. In addition, hybrid genes containing the human TK promoter linked to the bacterial neomycin resistance gene were induced by SV40 in stably transfected cells, indicating that promoter sequences are sufficient to confer viral regulation. Analysis of human TK promoter deletion mutants indicated that sequences localized between -67 and +30 bp relative to the transcriptional initiation site are sufficient to confer regulation on SV40-infected cells. These sequence elements are distinct from those required for serum induction, which were previously localized to the region between -135 and -67. These results suggest that SV40 activates novel cellular pathways that are not activated by serum stimulation of quiescent cells.

A microwell cytotoxicity assay using Artemia salina (brine shrimp).

A new microplate assay for cytotoxicity testing using A. salina has been developed and shown to give results comparable to a previously published test-tube method. The assay reliably detected all of the compounds toxic to KB cells in a series of 21 pharmacologically active agents, except for two which require metabolic activation in man. Four quassinoids with cytotoxic and antiplasmodial activity were also toxic to the brine shrimp while quassin itself was inactive in all three systems. It is proposed that this assay provides a convenient means by which the presence of cytotoxic quassinoids may be detected during the fractionation of plant extracts.

Quassinoids exhibit greater selectivity against Plasmodium falciparum than against Entamoeba histolytica, Giardia intestinalis or Toxoplasma gondii in vitro.

The in vitro activities of a series of quassinoids against Plasmodium falciparum, Entamoeba histolytica, Giardia intestinalis and Toxoplasma gondii have been compared with their in vitro cytotoxic effects against KB cells (human epidermoid carcinoma of the nasopharynx). All of the compounds tested were more toxic to KB cells than to G. intestinalis, but four (ailanthinone, bruceine D, brusatol and glaucarubinone) were slightly less toxic to KB cells than to E. histolytica. Glaucarubinone was similarly more toxic to intracellular T. gondii than to KB cells but ailanthinone was more selective (36 times more toxic to T. gondii than to KB cells). All of the compounds were more toxic to P. falciparum than to KB cells; the most selective quassinoids--glaucarubinone, bruceine D, ailanthinone and brusatol--were found to have toxicity/activity ratios of 285, 76, 48 and 32 respectively. These results suggest that quassinoids have a selective action on P. falciparum. Further studies to elucidate the basis for this are in progress.

Methods, statistical features, and baseline results of a standardized, multicentered ophthalmologic surgical trial: the Silicone Study.

This article describes the trial design and baseline results for the Silicone Study, a multicenter, randomized surgical trial designed to compare the effectiveness of silicone fluid versus long-acting gas in the treatment of proliferative vitreoretinopathy (PVR). Design features include (1) standardization of the surgical protocol to reduce intersurgeon variability, (2) formulation of a PVR clinical classification system relevant to modern vitreoretinal surgery, and (3) creation of a photographic protocol to document PVR pathology. Statistical issues affecting the analysis of the outcome data include (1) the addition of a second group of patients with more severely diseased eyes after the trial began, (2) the change to a different long-acting gas during the course of the trial, and (3) recurrent retinal detachments that require reoperations with the randomized treatment, and, in some instances, a crossover from the randomized to the alternate treatment. Demographic and baseline ocular characteristics are presented for the two groups under study.